Ezrin is overexpressed in a wide range of metastatic cancer types, but relatively little is known about what role Ezrin plays in promoting the metastatic phenotype. It is unclear why overexpression promotes metastasis due in part to our lack of understanding of how Ezrin is controlled and regulated. Connections have already been described that link the RhoA signaling pathway, which is also associated with metastatic phenotypes, to Ezrin activation. When Ezrin is activated it translocates to the interface between the plasma membrane and the actin cytoskeleton, where Ezrin acts as a cross linker. RhoA signaling also initiates protein activation cascades which cause the cell to produce force. This combination of RhoA force production and Ezrin's role as a plasma membrane and actin cytoskeleton cross linker suggests that Ezrin acts to reinforce RhoA generated intra- cellular forces at the plasma membrane. In addition, RhoA also interacts with RhoGDI and PKA, which can be activated by Ezrin at the plasma membrane. The goal of this proposal is to determine the role of Ezrin in mediating cell generated force at the plasma membrane and actin cytoskeleton. In Specific Aim 1, we will use a newly developed optically-based biosensor that measures tension across Ezrin in live cells to investigate how Ezrin loading is controlled. We use a series of drug treatments designed to perturb how Ezrin connects to the plasma membrane and cytoskeleton to determine when Ezrin is under tension. To determine how Ezrin loading is controlled by cell morphology and movement, we will also use micropatterning techniques control the shape, sizes and migration behavior of individual cells, while tracking Ezrin loading. In Specific Aim 2, we will investigate the connection between Ezrin and motility, through the RhoA signaling pathway. We will use an Ezrin knockdown cell line to test how the RhoA signaling cascade is affected through RhoGDI and PKA by the removal of Ezrin. To further our understanding of this system, we will also measure Ezrin loading as it compares to the activity of RhoA and PKA, providing a novel view into this complex system. The results of these aims will contribute to our understanding of how metastatic cancer cells migrate, potentially leading to the development of therapeutic treatments targeted at metastatic motility. This fellowship will provide me with new skills to allow my career to develop as an independent scientist, while also improving our understanding of an understudied aspect of metastasis.